Abstract
Rapid and extensive biliary excretion of [d-penicillamine2,5]enkephalin (DPDPE) in rats as the unchanged peptide suggests that multiple transport proteins may be involved in the hepatobiliary disposition of this zwitterionic peptide. Although DPDPE is a P-glycoprotein substrate, the role of other transport proteins in the hepatic clearance of DPDPE has not been established. Furthermore, the ability of various experimental approaches to quantitate the contribution of a specific hepatic uptake or excretion process when multiple transport systems are involved has not been addressed. 3H-DPDPE uptake in suspended Wistar rat hepatocytes was primarily (>95%) due to temperature-dependent transport mechanisms; similar results were obtained in suspended hepatocytes from Mrp2-deficient (TR-) rats. Pharmacokinetic modeling revealed that saturable and linear processes were involved in 3H-DPDPE uptake in hepatocytes. The use of transport modulators suggested that hepatic uptake of 3H-DPDPE was mediated by Oatp1a1, Oatp1a4, and likely Oatp1b2. Accumulation of 3H-DPDPE in sandwich-cultured (SC) hepatocytes was rapid; uptake of 3H-DPDPE in SC rat hepatocytes from control and TR- rats was similar. However, the biliary excretion index and biliary clearance decreased by 83 and 85%, respectively, in TR- SC rat hepatocytes, indicating that DPDPE is an Mrp2 substrate. Rate constants for uptake and excretion of 3H-DPDPE in SC rat hepatocytes were determined by pharmacokinetic modeling; data were consistent with basolateral excretion of 3H-DPDPE from the hepatocyte. These results demonstrate the complexities of hepatobiliary disposition when multiple transport mechanisms are involved for a given substrate and emphasize the necessity of multi-experimental approaches for the comprehensive resolution of these processes.
Footnotes
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This research was supported by National Institutes of Health Grant R01 GM41935.
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Kim L. R. Brouwer and Gary M. Pollack are co-founders of Qualyst, Inc. and members of the Scientific Advisory Board for Qualyst, Inc.; Qualyst Inc. has exclusively licensed the sandwich-cultured hepatocyte technology for quantification of biliary excretion (B-CLEAR).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.104.001420.
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ABBREVIATIONS: DPDPE, [d-penicillamine2,5]enkephalin; Oatp1a1, rat organic anion-transporting polypeptide 1a1 (formerly Oatp1); Oatp1a4, rat organic anion-transporting polypeptide 1a4 (formerly Oatp2); Oatp1b2, rat organic anion-transporting polypeptide 1b2 (formerly Oatp4); OATP1B1, human organic anion-transporting polypeptide 1B1 (formerly OATPC); OATP1B3, human organic anion-transporting polypeptide 1B3 (formerly OATP8); Bcrp, breast cancer resistance protein; Mrp2, multidrug resistance-associated protein 2; TR- rats, Mrp2-deficient rats; P-gp, P-glycoprotein; Mdr1a/b, multidrug resistance protein 1a/b; SC, sandwich-cultured; BEI, biliary excretion index; DIG, digoxin; BSP, bromosulfophthalein; DELT II, deltorphin II; TEA, tetraethylammonium; PAH, p-aminohippurate; BC, bile canaliculi; TBS-T, Tris-buffered saline containing 0.3% Tween 20; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution.
- Received July 10, 2004.
- Accepted November 3, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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